The invention relates to the use of dielectrophoresis in neutral particle classification, i.e., characterization and sorting. More particularly, an apparatus and method to produce continuous separation of neutral particle mixtures by the use of dielectrophoresis is disclosed. Further, the apparatus and method are particularly useful for the classification, i.e., characterization and separation, of living cells and their parts.
The need exists for methods of characterizing cells and separating out pure populations of cells from mixtures of cells. Cells which exhibit different functions and stages of differentiation must be separated from one another to facilitate the study of some of the molecular mechanisms involved in cell specialization, cytotoxicity and transformation. Diagnosis of diseased states and use of isolated cells for immunotherapy or transfusion purposes are areas where practical benefits of cell separation techniques may be attainable. It is of importance, for example, in the characterization of basic processes of cell differentiation and regulation, or of abnormalities. Looking ahead, it is not difficult to foresee that cell separations may play a vital roll in the diagnosis and treatment of disease. The problems of cell separation have received considerable attention in the past, resulting in the development of numerous separation methods. The techniques of electrophoresis and sedimentation were recently reviewed by Mel and Ross. 8 Quartr. Rev. Biophys. 421 (1975). Mel and Ross discussed sedimentation-centrifugation and electrophoresis. Methods using spectrophotometric signals on streams of cells to evoke a later "electrostatic" deflection of the droplet containing the labeled cell have been devised and offer considerable promise. Arndt-Jovin and Jovin, 7 Ann. Rev. of Biophys. Bio-eng. 527 (1978); Owen, 22 Biophys. J. 171 (1978). Fluorescence-activated cell sorting has been described by van Dilla. 163 Science 1213 (1969). A particularly interesting technique using high gradient magnetic fields has been described by Molday and by Kronick. Molday, 268 Nature (London) 437 (1977); Kronick, 200 Science 1074 (1978).
Dielectrophoresis has been defined as the motion of a neutral particle due to the action of a nonuniform electric field on its permanent or induced dipole moment. When a particle is introduced into a system with a nonuniform electric field, the field induces a dipole in that particle. The divergent nonuniform nature of the field results in one end of the dipole being in a region of higher field strength than the other. The effect is that the dipole is pulled in the direction of the increasing field.
Nonuniform electric fields can induce translational and rotational motions of cells in suspension. These motions can be used to characterize and usefully separate living cells and their parts. The nonuniform field acts by aligning or inducing a dipole moment in the cell. The cell is then impelled by the field nonuniformity, usually towards the region of greatest field intensity.
The force created is known as the dielectrophoretic force, and the resulting motion dielectrophoresis. In the event the cell being acted upon is suspended in a polarizable medium, the net polarization of the whole may be such as to evoke a dielectrophoretic force in favor of pushing the body either into or away from the region of higher field intensity. The cell experiences "positive" dielectrophoresis when it is forced into the region of higher field intensity; "negative" dielectrophoresis results when the cell is pushed away from the region of higher field intensity.
It is well-known that a neutral particle, when subjected to the influence of a nonuniform, time varying (AC) electric field, may exhibit one of the following behaviors:
(1) Positive dielectrophoresis, i.e. attraction toward the region of high field intensity; PA0 (2) Negative dielectrophoresis, i.e. repulsion toward the region of lower field intensity; or PA0 (3) Zeresis, i.e. no net displacement. PA0 (1) avoidance of local overheating, as by the application of high local current in Joule Heating; PA0 (2) minimization of dead-flow pockets; PA0 (3) minimization of turbulence; PA0 (4) avoidance of cell damage as by grinding, adhesion or heat, and by electrolytic or chemical changes, as of pH; PA0 (5) maintenance of the individuality of cells.
These processes arise from the following sequence of phenomena. The electric field induces a charge separation or dipole in the neutral particle. The resultant dipole consisting of equal numbers of slightly separated positive and negative charges now experiences a net force upon it because of the nonuniformity of the electric field. One or the other of the charge sets will be in a weaker electric field. Since the force upon a charge is exactly dependent upon the amount of charge, and upon the local field acting upon that charge, it will be seen that a net force arises upon the particle, despite the fact that it is neutral overall and has no excess charge of either type. The same considerations apply to the supporting fluid medium. The net of these dielectrophoretic forces upon the particle and its supporting medium acts to impel the particle toward the stronger field in positive dielectrophoresis. If, on the other hand, the net force upon the particle and medium is such as to impel the particle toward the region of weaker field, negative dielectrophoresis results.
In electrophoresis, the field induced motion of charged particles, the direction of the force is dependent upon the sign of the charge and upon the direction of the field. However, in dielectrophoresis, the force depends upon the square of the field intensity, and is independent of the direction of the field. For this reason, dielectrophoresis works well in AC fields.
For a particle to experience either positive or negative dielectrophoresis it must be subject to a divergent electric field. In practice, nonuniform electric fields can be realized using any of many electrode geometries. Pohl, 5 J. Electrostatics 337 (1978) discussing these geometries is incorporated by reference herein. However, in order to obtain sharp cell separations, it is considered that the "isomotive" field is the most appropriate, as it offers a constancy of dielectrophoretic force operating over a relatively wide region. Theoretical considerations in the design of "isomotive" cell electrodes are discussed in U.S. Pat. No. 3,162,592, incorporated by reference herein.
Among the factors which must be given close attention during the successful separation of living cells by continuous dielectrophoresis are:
The need existed in the prior art for an apparatus and method meeting the above factors. Further, the need existed for an apparatus and method to classify large quantities of neutral particles in relatively short periods of time.